The proposed renewal project focuses on mechanistic aspects of antiviral RNA silencing using the Arabidopsis model. The genetic, genomic and technical aspects of this model have proven exceptionally useful in revealing RNA silencing functions that limit virus infection at the cell-autonomous and cellnonautonomous levels, as well as silencing functions that govern stress responses, development, and repressive chromatin. In the current project period, Arabidopsis was used to identify the roles of cellular and viral factors, such as virus-encoded suppressors of RNA silencing, during antiviral defense and counterdefensive processes. This, and work from several other groups, led to conceptualization of a threephase model for antiviral silencing in Arabidopsis. This model describes events occurring during the initial targeting phase, the siRNA amplification phase and the systemic silencing phase. This proposed research seeks to test key elements of each phase of the model. The three Aims are summarized as follows: ? Aim 1 - Understand silencing events during the initial targeting phase. New high-throughput sequencing technology will be used to analyze the genetic requirements for initial targeting of viral genomes by DCL factors during early stages of infection, and test the hypothesis that initial targeting yields primarily (+)-sense siRNA that seed the subsequent amplification step. ? Aim 2 - Understand silencing events during the amplification phase. The genetic requirements and detailed accumulation patterns of siRNA formed during the RDR6-dependent amplification phase will be determined, and the small RNA populations that interact with a silencing suppressor during antiviral silencing will be identified. ? Aim 3 - Understand silencing events during the systemic phase. The hypothesis that viral silencing suppressors function in vascular cells to inhibit cell-nonautonomous, DCL4-dependent systemic signals that limit virus invasiveness will be tested.